Specific antagonists for glucose-dependent insulinotropic polypeptide (GIP)

ABSTRACT

In one embodiment, this invention provides an antagonist of glucose-dependent insulinotropic polypeptide (GIP) consisting essentially of a 24 amino acid polypeptide corresponding to positions 7-30 of the sequence of GIP. In another embodiment, this invention provides a method of preventing and treating obesity and non-insulin dependent diabetes mellitus (Type II) in a patient comprising administering to the patient an antagonist of glucose-dependent insulinotropic polypeptide (GIP). In yet another embodiment, this invention provides a method of improving glucose tolerance in a mammal comprising administering to the mammal an antagonist of glucose-dependent insulinotropic polypeptide (GIP).

The work leading to this invention was supported in part by Grant Nos.DK08753 and RO1DK48042 from the National Institutes of Health. The U.S.Government may have certain rights to this invention.

FIELD OF THE INVENTION

This invention is directed to specific antagonists of glucose-dependentinsulinotropic polypeptide (GIP). This invention is also directed totreatment of non-insulin dependent diabetes through increasing glucosetolerance without requirement for increased serum insulin, the treatmentof obesity by the administration of a GIP antagonist, the development ofnonpeptide GIP antagonist compounds, and compositions.

BACKGROUND

Insulin release induced by the ingestion of glucose and other nutrientsis due part to both hormonal and neural factors (Creutzfeldt, et al.,1985, Diabetologia 28:565-573). Several gastrointestinal regulatorypeptides have been proposed as incretins, the substance(s) believed tomediate the enteroinsular axis and that may play a physiological role inmaintaining glucose homeostasis (Unger, et al., 1969, Arch. Intern. Med,123:261-266; Ebert R., et al. 1987, Diab. Metab. Rev., 3:1-16; Dupré J.,1991, “The Endocrine Pancreas.” Raven Press, New York, p 253). Amongthese candidates, only glucose-dependent insulinotropic polypeptide(GIP) and glucagon like peptide-1 (7-36)(GLP-1) appear to fulfill therequirements to be considered physiological stimulants of postprandialinsulin release (Dupré, et al. 1973, J. Clin. Endocrinol. Metab.,37:826-828; Nauck, et al., 1989; J. Clin. Endocrinol. Metab.,69:6540662; Kreymann, et al. 1987, Lancet, 2:1300-1304; Mojsov, et al.,1987, J. Clin. Invest., 79:616-619).

Following oral glucose administration, serum GIP levels increase severalfold (see Cleator, et al., 1975, Am. J. Surg., 130:128-135; Nauck, etal. 1986, J. Clin. Endocrinol. Metab., 63:492-498; Nauck, et al., 1986,Diabetologia, 29:46-52; Salera, et al., 1983, Metabolism, 32:21-24;Kreymann, et al., 1987, Lancet, 2:1300-1304), and although the incrementin plasma GLP-1 concentration in response to glucose is alsosignificant, it is far smaller in magnitude (Kreymann, et al., 1987,Lancet, 2:1300-1304; Ørskov, et al., 1987, Scand. J. Clin. Lab. Invest.,47:165-174; Ørskov, et al., 1991, J. Clin. Invest., 87:415-423; Shuster,et al., 1988, Mayo Clin. Proc., 63:794-800). In human volunteers, Naucket al. (1993, J. Clin. Endocrinol. Metab., 76:912-917) showed that GIPwas a major contributor in the incretin effect after oral glucose,whereas GLP-1 appeared to play a major role. Shuster et al. (1988) alsosuggested that GIP was the most important, but not the sole, mediator ofthe incretin effect in humans.

Some studies have demonstrated that GIP and GLP-1 are equally potent intheir capacity to stimulate insulin release (Schmid, et al., 1990, Z.Gastroenterol., 28:280-284; Suzuki, et al., 1990, Diabetes,39:1320-1325), whereas others have suggested that GLP-1 possessesgreater insulinotropic properties (Siegel, et al. 1992, Eur. J. Clin.Invest. 22:154-157; Shima, et al. 1988, Regul. Pept., 22:245-252).Recently, using a putative specific antagonist to the GLP-1 receptor,exendin (9-39), Wang et al. have demonstrated that exenden reducedpostprandial insulin release by 48% and thus concluded that GLP-1 mightcontribute substantially to postprandial stimulation of insulinsecretion (Wang, et al. 1995, J. Clin. Invest., 95:417-421). More recentstudies, however, have shown that exendin might also displace GIPbinding from its receptor and thereby reduce GIP-stimulated cyclicadenosine monophosphate (cAMP) generation (Wheeler, et al. 1995,Endocrinology, 136:4629-4639; Gremlich, et al. 1995, Diabetes,44:1202-1208). Therefore, the antagonist properties of exendin (9-39)might not be limited to GLP-1.

The availability of a GIP-specific receptor antagonist would beinvaluable for determining the precise roles of these peptides inmediating postprandial insulin secretion.

SUMMARY OF THE INVENTION

It is an object of this invention to provide specific antagonists ofglucose-dependent insulinotropic polypeptide (GIP).

It is another object of this invention to provide alternative methodsfor treatment of non-insulin dependent diabetes which increase glucosetolerance without requirement for increased serum insulin, for treatmentof obesity with a GIP antagonist which inhibits, blocks or reducesglucose absorption from the intestine of an animal, and for developmentof nonpeptide GIP antagonist compounds.

In one embodiment, this invention provides an antagonist ofglucose-dependent insulinotropic polypeptide (GIP) consistingessentially of a 24-amino acid polypeptide corresponding to positions7-30 of the sequence of GIP.

In another embodiment, this invention provides a method of treatingnon-insulin dependent diabetes mellitus in a patient comprisingadministering to the patient an antagonist of glucose-dependentinsulinotropic polypeptide (GIP).

In yet another embodiment, this invention provides a method of improvingglucose tolerance in a mammal comprising administering to the mammal anantagonist of glucose-dependent insulinotropic polypeptide (GIP).

Using a reporter L-cell line stably transfected with rat GIP receptorcDNA (LGIPR2), the inventors have identified a fragment of GIP [GIP(7-30)-NH₂] as a specific GIP receptor antagonist. This antagonist(referred to as ANTGIP) inhibited GIP-stimulated intracellular cAMPproduction in vitro, and ANTGIP competed with GIP for binding tocellular receptors, but did not complete with GLP-1. ANTGIP inhibitedthe GIP-dependent release of insulin in vivo, but ANTGIP had no effecton glucose-, GLP-1-, GIP-, and arginine-induced insulin release inanesthetized rats. In conscious rats, ANTGIP inhibited postprandialinsulin release, without significantly affecting the serum glucoseconcentration. However, despite its inhibiting effect on insulinrelease, ANTGIP has been discovered to enhance glucose tolerance in anoral glucose tolerance test.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show cAMP-dependent β-galactosidase production by LGIPR2cells in the presence of GIP or various GIP fragments.

FIG. 2 shows dose-dependent inhibition of ANTGIP on GIP-includedcAMP-dependent β-galactosidase production in LGIPR2 cells.

FIG. 3 shows competition of ¹²⁵I-GIP and ¹²⁵I GLP-1 (inset) binding byGIP, GLP-1 and ANTGIP.

FIG. 4 shows plasma insulin concentrations (±SE) in fasted anesthetizedrats after 30 min of GIP, ANTGIP, or 0.9 NaCl infusion.

FIG. 5 shows plasma insulin concentrations (±SE) in fasted anesthetizedrats after a 30-min infusion of GLP-1 (0.4 nmol/kg), glucose (0.8 g/kg),or arginine (375 mg/kg) with (open bars) or without (solid bars) ANTGIP(100 nmol/kg) (n=6 for each group).

FIG. 6 shows postprandial plasma insulin and serum glucose levels (±SE)in conscious trained rats.

FIG. 7 shows plasma insulin level following oral glucose administrationto rats with or without ANTGIP injection.

FIG. 8 shows plasma glucose level following oral glucose administrationto rats with and without ANTGIP injection.

FIG. 9 shows the effects of the GIP receptor antagonist, ANTGIIP, on theabsorption of free D-glucose from the lumen of the jejunal test segment.

DETAILED DESCRIPTION OF THE INVENTION

Glucose-dependent insulinotropic polypeptide (GIP) is 42-amino acidhormone that was originally described as a inhibitor of acid secretion.More recently, however, it has been shown to be potent stimulant for therelease of insulin from the endocrine pancreas.

The inventors have confirmed previous studies (Rossowski, et al., 1992,Regul. Pep., 39:9-17) indicating that truncated GIP [GIP (1-30)-NH₂]might be one of the biologically active forms of mature GIP. As shown inFIG. 1, GIP (1-30)-NH₂ was nearly equipotent to GIP (1-42) instimulating cAMP dependent β-galactosidase production in LGIPR2 cells.These findings are consistent with the observations of Wheeler, et al.(1995), reported that both GIP(1-42) and GIP(1-30) exhibited similarstimulatory properties for cAMP production in COS-7 cell transientlyexpressing GIP receptor cDNA. Moreover, Kieffer et al. (1993, Can. J.Physiol. Pharmacol., 71:917-922) found that GIP (1-30) competitivelyinhibited binding of GIP (1-42) to the GIP receptor in βTC3 cells. Thesedata suggest the possibility of cellular processing of GIP (1-42) toyield biologically-active α-amidated GIP (1-30).

Physiological Effects of GIP Antagonists

Insulin release induced by the ingestion of glucose and other nutrientsis due in part to both hormonal and neural factors (see, e.g.,Creutzfeldt, et al., 1985). Although a number of gastrointestinalregulatory peptides have been proposed as putative incretins; GIP andGLP-1 are the most likely physiological insulinotropic peptides.Although both GIP and GLP-1 possess significant insulinotropicproperties, controversy exists regarding their relative physiologicalroles in stimulating insulin release.

Using a GLP-1 receptor antagonist exendin (9-39), Wang et al. (1995)detected a 50% decrease in postprandial insulin secretion inexendin-treated rats. Administration of exendin also reduced 70% ofinsulin release following intraduodenal glucose infusion (Kolligs, etal., 1995, Diabetes, 44:16-19). Recent studies, however, havedemonstrated that exendin also displaced GIP binding from its receptor,and inhibits cAMP generation in response to GIP stimulation (Wheeler, etal. 1995; Gremlich, et al. 1995). Therefore, the antagonist propertiesof exendin do not appear to be GLP-1 specific.

Successful synthesis by the present inventors of a specific GIP receptorantagonist greatly facilitates investigation of the relativecontribution of GIP in mediating the enteroinsular axis. The GIPfragment ANTGIP [GIP (7-30)-NH₂] specifically inhibits variousGIP-dependent effects. In LGIPR2 cells, ANTGIP inhibited the cAMPresponse to GIP in a concentration-dependent manner (see FIG. 2), and inβTC3 cells, the antagonist displaced GIP binding from its receptor (seeFIG. 3). Furthermore, ANTGIP completely abolished the insulinotropicproperties of GIP in fasted anesthetized rats, while not affectingGLP-1, glucose-, or arginine-stimulated insulin release indicating thatthis antagonist is GIP-specific. ANTGIP alone demonstrated nostimulatory effect on insulin release or cAMP generation in eitherintact rats or LGIPR2 cells, indicating the absence of any agonistproperties. Studies demonstrated that even at a concentration as high as10⁻⁴ M, ANTGIP did not stimulate a detectable increase in cAMP-dependentβ-galactosidase level in LGIPR2 cells.

The inventors have observed a 72% decrease in postprandial insulinrelease in response to the administration of ANTGIP to rats. ANTGIP didnot affect GLP-1 binding to its receptor, and the insulinotropic effectof GLP-1 is preserved in vivo in the presence of ANTGIP. Furthermore,postprandial GLP-1 levels were not affected by ANTGIP. These findingsare consistent with a dominant role for GLP in mediating theenteroinsular axis.

Wang et al. demonstrated an approximate 50% reduction in postprandialinsulin levels in exendin-treated rats, whereas plasma glucose levelsincreased minimally from 7.5 to 8.7 mmol/l. The physiologicalsignificance of this minor increment in glucose level was not clear toWang, et al. The inventors found that serum glucose concentrationsremained largely unchanged despite a marked decrease in serum insulinlevels in ANTGIP-treated rats. The results of the present study areconsistent with the notion that insulin is not the sole mediator ofglucose homeostasis, but that glucose maintenance is dependent onnumerous neurohumoral factors. These factors include hormones, such aspancreatic glucagon, cortisol, and growth hormone, and physiologicalevents, including peripheral and hepatic glucose uptake.

The results of the present studies demonstrate that GIP (7-30)-NH₂ is aspecific receptor antagonist of naturally occurring GIP. GIP (7-30)-NH₂inhibits GIP-induced cAMP generation and insulin release, but does notaffect the insulinotropic effects of other secretagogues such asglucose, arginine, and GLP-1. Furthermore, circulating insulin levelsdecreased by 72% in response to the concomitant administration of GIP(7-30)-NH₂ to chow-fed rats, indicating that GIP plays a dominant rolein mediating postprandial insulin secretion.

Strikingly, although GIP (7-30)-NH₂ reverses the insulin stimulatoryproperties of the parent compound, when the GIP antagonist wasadministered to rats (injected intraperitoneally), oral glucosetolerance was improved: a significant decrease in serum glucose levelswas detected at all time points in all rats. In addition, plasma insulinlevels were also diminished in these same rats. These results aresurprising—with the decrease in insulin release, one would expect anincrease in serum glucose. However, GIP has several other peripheraleffects which may include an affect of GIP on peripheral glucoseutilization, and the decrease in serum glucose levels seen with GIPmight be due to such an effect.

The effect of GIP antagonists on serum glucose levels in the absence ofincreased serum insulin suggests their use in patients with noninsulindependent diabetes mellitus (NIDDM). With the aging of the United Statespopulation, an increase in the number of cases of NIDDM has beenpredicted. In the past forty years, very few new forms of therapy forthis most prevalent disease have been developed. GIP antagonists enhancetolerance to oral glucose, as demonstrated herein, and thereforetreatment of NIDDM patients with these compounds is indicated.

GIP Antagonists

A GIP antagonist according to this invention is any composition whichinterferes with biological action of GIP. Such compositions includeantibodies specific for either GIP or GIP receptors, antisense RNA whichhybridizes with mRNA encoding GIP or GIP receptor, or other geneticcontrols which knock out expression of GIP or GIP receptor. GIPantagonists also include peptides or other small molecules which bind tothe GIP receptor and block the cAMP response to GIP. Suitable assays forantagonist activity are exemplified in Examples 1 and 2 below:

As described herein (see Example 1 below), the inventors have nowdiscovered a polypeptide fragment of GIP that is a specific GIP receptorantagonist. While the 30-amino acid N-terminal fragment [GIP (1-30)-NH₂]was as effective in stimulating cAMP increase through GIP receptors asthe parent hormone, a fragment missing the most N-terminal six aminoacids [GIP (7-30)-NH₂] did not stimulate cAMP release in the samesystem. Thus, the N-terminal hexamer appears to be important forfunctional GIP signaling. GIP fragments missing the N-terminal 15 aminoacids (e.g., GIP (16-30)-NH₂) did not mimic GIP, but neither did theyinhibit GIP-dependent effects. Thus, the segment from amino acids 7-15appears to be especially important in signaling through the GIPreceptor. Fragment GIP (10-30)-NH₂ was less effective as an antagonist,but retained some ability to affect GIP receptor activation, asindicated by partial agonist activity. Thus, peptide antagonists wouldappear to require the segment from amino acids 7-9 of the GIP sequence,and some or all of the amino acids from 10-30 or effective alternativeamino acids thereto are likely to promote binding to the receptor. Itshould therefore be understood by those of skill in this art that thepresent invention contemplates any polypeptide sequence whicheffectively prevents GIP activation of its native receptor, such as thesequence containing amino acids in positions 7-30 of the sequence of theGIP sequence and polypeptides based upon sequences containing aminoacids in positions 7-30 of the sequence of the GIP that includeadditional, deleted or alternative amino acids to form effective GIPpolypeptide antagonist. Polypeptides based on this sequence may bedesigned for use as GIP antagonists according to this invention by theskilled artisan, who will routinely confirm that the resultant peptidesexhibit antagonist function by testing the peptides in in vitro and invivo assays such as those described in Examples 1 and 3-5 below.

Immunologic components specific for GIP or GIP receptors can be employedas GIP antagonists. Such antagonists include with specific monoclonalantibodies (either naked or conjugated to cytotoxic agents) or specificactivated cytotoxic immune cells. Such antibodies or immune cells may begenerated as reagents outside the body, or may be generated inside thebody by vaccines which target GIP or GIP receptors.

Antibodies which are specifically reactive with GIP or the hormonebinding domain of GIP receptor, or antigenic recombinant peptidefragments of either of those proteins, may be obtained in a number ofways which will be readily apparent to those skilled in the art. Theknown sequences of GIP (see Takeda, et al. 1987, Proc. Natl. Acad. SciUSA, B84:7005-7008, and Genbank Accession No. M18185), and GIP receptor(see Bonner, T. I., and Usdin, T. B., 1995, Genbank Accession No U39231)can be used in conjunction with standard recombinant DNA technology toproduce the desired antigenic peptides in recombinant systems (see,e.g., Sambrook et al.). Antigenic fragments of GIP or GIP receptor canbe injected into an animal as a immunogen to elicit polyclonal antibodyproduction. Purification of the antibodies can be accomplished byselective binding from the serum, for instance by using cellstransformed with DNA sequence encoding the respective proteins. Theresultant polyclonal antisera may be used directly or may be purifiedby, for example, affinity absorption using recombinantly producedprotein coupled to an insoluble support.

In another alternative, monoclonal antibodies specificallyimmunoreactive with either GIP or the hormone binding domain of GIPreceptor may be prepared according to well known methods (See, e.g.,Kohler and Milstein, 1976, Eur. J. Immunol., 6:611), using the proteinsor antigenic fragments described above as immunogen(s), using them forselection or using them for both functions. These and other methods forpreparing antibodies or immune cells that are specificallyimmunoreactive with GIP or GIP receptor are easily within the skill ofthe ordinary worker in the art.

Immunogenic compositions according to this invention for use in activeimmunotherapy include recombinant antigenic fragments of GIP or GIPreceptor prepared as described above and expression vectors(particularly recombinant viral vectors) which express antigenicfragments of GIP or GIP receptor. Such expression vectors can beprepared as described in Baschang, et al., U.S. Pat. No. 4,446,128,incorporated herein by reference, or Axel, et al., Pastan, et al., orDavis, et al., using the known sequences of GIP or GIP receptor.

Still another GIP antagonist according to this invention is anexpression vector containing an antisense sequence corresponding to allor part of an mRNA sequence encoding GIP or GIP receptor, inserted inopposite orientation into the vector after a promoter. As a result, theinserted DNA will be transcribed to produce an RNA which iscomplementary to and capable of binding or hybridizing to the mRNA. Uponbinding to the GIP or GIP receptor mRNA, translation of the mRNA isprevented, and consequently the protein coded for by the mRNA is notproduced. Suitable antisense sequences can be readily selected by theskilled artisan from the sequences of GIP or GIP receptor cited above.Production and use of antisense expression vectors is described in moredetail in U.S. Pat. No. 5,107,065 and U.S. Pat. No. 5,190,931, both ofwhich are incorporated herein by reference.

Alternative materials within the contemplation of the skilled artisanwhich function as antagonists of GIP in the procedures described inExamples 1 and 3-5 below may also be used in the therapeutic methodsaccording to this invention.

Therapeutic Use of GIP Antagonists

GIP (7-30)-NH₂ acts as a receptor antagonist of GIP, but also improvesglucose tolerance contrary to the expected consequence of blockingGIP-dependent insulin secretion. In addition, a GIP receptor antagonistin accordance with the present invention inhibits, blocks or reducesglucose absorption from the intestine of an animal. In accordance withthis observation, therapeutic compositions containing GIP antagonistsmay be used in patients with noninsulin dependent diabetes mellitus(NIDDM) to improve tolerance to oral glucose or in animals, such ashumans, to prevent, inhibit or reduce obesity by inhibiting, blocking orreducing glucose absorption from the intestine of the animal, asdemonstrated herein.

Therapeutic compositions according to this invention are preferablyformulated in pharmaceutical compositions containing one or more GIPantagonists and a pharmaceutically acceptable carrier. Thepharmaceutical composition may contain other components so long as theother components do not reduce the effectiveness of the GIP antagonistaccording to this invention so much that the therapy is negated.Examples of such components include sweetening, flavoring, coloring,dispersing, disintegrating, binding, granulating, suspending, wetting,preservative and demulcent agents and the like. Pharmaceuticallyacceptable carriers are well known, and one skilled in thepharmaceutical art can easily select carriers suitable for particularroutes for administration (Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., 1985).

Also in accordance with the present invention, the GIP receptorantagonist of the present invention may be lyophilized using standardtechniques known to those in this art. The lyophilized GIP receptorantagonists may then be reconstituted with, for example, suitablediluents such as normal saline, sterile water, glacial acetic acid,sodium acetate, combinations thereof and the like. The reconstituted GIPreceptor antagonists in accordance with the present invention may beadministered parenterally or orally and may further includepreservatives or other acceptable inert components as mentionedhereinbefore.

The pharmaceutical compositions containing any of the GIP antagonistsaccording to this invention may be administered by parenteral(subcutaneously, intramuscularly, intravenously, intraperitoneally,intrapleurally, intravesicularly or intrathecally, topical, oral,rectal, or nasal route, as necessitated by choice of drug and disease.The dose used in a particular formulation or application will bedetermined by the requirements of the particular state of disease andthe constraints imposed by the characteristics of capacities of thecarrier materials. The concentrations of the active agent inpharmaceutically acceptable carriers may range from 0.1 nM to 100 μM.The compositions described above may be combined or used together or incoordination with another therapeutic substance.

Dose will depend on a variety of factors, including the therapeuticindex of the drugs, disease type, patient age, patient weight, andtolerance of toxicity. Dose will generally be chosen to achieve serumconcentrations from about 0.1 μg/ml to about 100 μg/ml. Preferably,initial dose levels will be selected based on their ability to achieveambient concentrations shown to be effective in in-vitro models, such asthat used to determine therapeutic index, and in-vivo models and inclinical trials, up to maximum tolerated levels. Standard clinicalprocedure prefers that chemotherapy be tailored to the individualpatient and the systemic concentration of the chemotherapeutic agent bemonitored regularly. The dose of a particular patient can be determinedby the skilled clinician using standard pharmacological approaches inview of the above factors. The response to treatment may be monitored byanalysis of blood or body fluid levels of the glucose or GIP or GIPantagonist according to this invention, measurement of activity if theantagonist or its levels in relevant tissues or monitoring disease stateof the patient. The skilled clinician will adjust the dose based on theresponse to treatment revealed by these measurements.

One approach to therapy of NIDDM is to introduce vector expressingantisense sequences to block expression of GIP and/or GIP receptor. Inone embodiment of this invention, a method is provided which comprisesobtaining a DNA expression vector containing a cDNA sequence having thesequence of human GIP or GIP receptor mRNA which is operably linked to apromoter such that it will be expressed in antisense orientation, andtransforming cells which express GIP or GIP receptor, respectively, withthe DNA vector. The expression vector material is generally produced byculture of recombinant or transfected cells and formulated in apharmacologically acceptable solution or suspension, which is usually aphysiologically-compatible aqueous solution, or in coated tablets,tablets, capsules, suppositories, inhalation aerosols, or ampules, asdescribed in the art, for example in U.S. Pat. No. 4,446,128,incorporated herein by reference.

The vector-containing composition is administered to a mammal exhibitingNIDDM in an amount sufficient to transect a substantial portion of thetarget cells of the mammal. Administration may be any suitable route,including oral, rectal, intranasal or by intravesicular (e.g. bladder)instillation or injection where injection may be, for example,transdermal, subcutaneous, intramuscular in intravenous. Preferably, theexpression vector is administered to the mammal so that the target cellsof the mammal are preferentially transfected. Determination of theamount to be administered will involve consideration of infectivity ofthe vector, transection efficiency in vitro, immune response of thepatient, etc. A typical initial dose for administration would be 10-1000micrograms when administered intravenously, intramuscularly,subcutaneously, intravesicularly, or in inhalation aerosol, 100 to 1000micrograms by mouth, 10⁵ to 10¹⁰ plaque forming units of a recombinantvector, although this amount may be adjusted by a clinician doing theadministration as commonly occurs in the administration of otherpharmacological agents. A single administration may usually besufficient to produce a therapeutic effect, but multiple administrationsmay be necessary to assure continued response over a substantial periodof time.

Further description of suitable methods of formulation andadministration according to this invention may be found in U.S. Pat.Nos. 4,592,002 and 4,920,209, which are incorporated herein by referencein their entireties.

The present invention also contemplates the use of the GIP antagonistsand/or its properties to develop nonpeptide compounds which exhibitantagonist properties similar to the GIP polypeptide antagonists asherein described using techniques known those versed in thepharmaceutical industry.

EXAMPLES

In order to facilitate a more complete understanding of the invention, anumber of Examples are provided below. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only.

Example 1 Effects of Various Peptide Fragments on cAMP Production

To define the biologically active region of GIP, the effects of severalpeptide fragments of GIP on stimulating cAMP-dependent β-galactosidaseproduction in LGIPR2 cells were examined. LGIPR2 cells are stablytransfected with a cAMP-dependent promoter from the VIP gene fused tothe bacterial lac Z gene. When intracellular cAMP increases within thesecells, lac Z gene transcription is activated, resulting in theaccumulation of its product, β-galactosidase. The measurement ofβ-galactosidase in this system provided a convenient, inexpensive, andnonradioactive method for detecting changes in the levels ofintracellular cAMP.

LGIPR2 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM)containing 4.5 g/L of glucose and 10% fetal calf serum. For each assay,10⁵ cells/well were seeded onto 24-well plates. After incubationovernight, peptides were added in various concentrations to the wells inthe absence of 3-isobutyl-methylxanthine (IBMX) for 4 h, at which timemaximal stimulation of β-galactosidase was determined. The medium wasthen removed and wells rinsed once with phosphate-buffered saline (PBS).The plates were then blotted briefly and frozen overnight at −70° C.,and, after the addition of chlorophenol red-β-D-galactopyranoside,accumulated β-galactosidase was detected using a colorimetric assay, asdescribed previously (Usdin, et al., 1993, Endocrinology,133:2861-2870).

Preliminary studies using LGIPR2 cells demonstrated that GIP(1-42)stimulated β-galactosidase production in a concentration-dependentmanner, with the maximum effect observed at 4 h with 10⁻⁸ M. Variouspeptide fragments of GIP, including GIP(21-30)NH₂, GIP (16-30)-NH₂, GIP(7-30)-NH₂, GIP (1-30)-NH₂, GIP (10-30)-NH₂, and GIP (31-44), weresynthesized at the Biopolymer Laboratory, Harvard Medical School, basedon previously published rat GIP cDNA sequence (Tseng, et al., 1993,Proc. Natl. Acad. Sci. USA, 90:1992-1996). LGIPR2 cells were incubatedin the presence of 10⁻M GIP or different GIP fragments for 4 h, andβ-galactosidase was measured as described herein and expressed inoptical density (O.D.) units. FIGS. 1A and 1B show cyclic AMP-dependentβ-galactosidase generation in LGIPR2 cells in response to incubationwith different fragments of GIP. Values are expressed as the mean ±SE ofquadruplicate measurements (*p<0.01, compared to control).

As demonstrated in FIG. 1A, 10⁻⁸ M GIP (1-30)-NH₂ stimulatedβ-galactosidase production to a similar degree, while none of the otherpeptide fragments tested, including GIP (7-30)-NH₂, GIP (16-30)-NH₂, GIP(21-30)-NH₂, and GIP (31-44), stimulated β-galactosidase generationabove control levels. Furthermore, no changes incAMP-dependent-β-galactosidase levels were detected when LGIPR2 cellswere incubated in the presence of higher concentrations of the smallerpeptide fragments.

To examine whether any of these fragments might serve as an antagonistto GIP, LGIPR2 cells were incubated with 10⁻⁸ M GIP (1-42) and one ofthe peptide fragments at two different concentrations (10⁻⁸ M or 10⁻⁶ M)for 4 h. LGIPR2 cells were cultured in the presence of 10⁻¹ M GIP andvarious concentrations of ANTGIP, as depicted on the horizontal axis ifFIG. 2. Values are expressed as the mean ±SE of quadruplicatemeasurements. Only GIP (7-30)-NH₂ (ANTGIP) was found to attenuate thecAMP stimulatory effects exhibited by GIP (1-42); the inhibition wasconcentration-dependent, with half-maximal inhibition occurring at 10⁻⁷M (FIG. 2).

FIG. 1B shows that peptide GIP (10-30)-NH₂ is an antagonist, albeit aweak one, as demonstrated by the reduction in GIP-stimulated β-gallevels when GIP (10-30)-NH₂ is present with GIP (1-42) compared to GIP(1-42) alone. On the other hand, GIP (10-30)-NH₂ also has agonistproperties, as demonstrated by β-gal level of 0.39 O.D. ±0.03 stimulatedby GIP (10-30)-NH₂ alone, compared to 0.95±0.04 for GIP (1-42).

Example 2 Receptor Binding Studies

Binding studies were performed in either LGIPR2 or βTC3 cells todetermine the relative affinities of GIP, ANTGIP, and GLP-1 for both GIPand GLP-1 receptors. GLP(7-37) and porcine GIP (5 μg each) wereiodinated by the chloramine-T method and were purified using C-18cartridges (Sep-Pak®, Millipore, Milford, Mass.) using an acetonitrilegradient of 30-45%. The specific activity of radiolabeled peptides was10-50 μCi/mg (Hunter, et al., 1962, Nature, 194:495-498; Kieffer, etal., 1993, Can. J. Physiol. Pharmacol., 71:917-922). Aliquots werelyophilized and reconstituted in assay buffer at 4° C. to aconcentration of 3×10⁵ cpm/100 μl. Binding studies was performed indesegrated LGIPR2 or βTC2 cells, the latter a generous gift from Dr. S.Efrat (Diabetes Center, Albert Einstein College of Medicine, New York).The βTC2 cell line originally arose in a lineage of transgenic miceexpressing an insulin promoted, SV40 T-antigen hybrid oncogene inpancreatic β-cells (Efrat, et al., 1988, Proc. Natl. Acad. Sci. U.S.A.,85:9037-9041) and has previously been demonstrated to be responsive toboth GIP and GLP (Kieffer, et al., 1993, Can. J. Physiol. Pharmacol.,71:917-922). The receptor binding buffer contained 138 mM NaCl, 5.6 mMKCl, 1.2 mM MgCl₂, 2.6 mM CaCl₂, 10 mM Hepes, 10 mM glucose, and 1%bovine serum albumin (BSA, fraction V, protease free, Sigma). Forbinding assays, LGIPR2 (GIP binding) or βTC3 (GLP-1 binding) cells werecultured in DMEM containing 4.5 g/L of glucose and 10% fetal bovineserum until 70% confluent. Cells were washed once with PBS and thenharvested with PBS-EDTA solution. βTC3 cells were then suspended inassay buffer at a density of 2×10⁶ cells/ml, and LGIPR2 cells were usedat a density of 2.5×10⁵ cells/ml. Binding was performed at roomtemperature in the presence of 3×10⁵ cpm/ml of [¹²⁵I]-GIP and -GLP.Nonsaturable binding was determined by the amount of radioactivityassociated with cells when incubated in the presence of unlabeled 10⁻⁶ MGIP, GLP, or 10⁻⁴ M ANTGIP. Specific binding was defined as thedifference between counts in the absence and presence of unlabeledpeptide. GIP binding was examined using LGIRP2 cells, and GLP-1 bindingwas assessed using βTC3 cells, and the results are shown in FIG. 3.Values are expressed as a percentage of maximum specific binding and arethe mean ±SE, with assays performed in duplicate.

GIP and ANTGIP displaced the binding of [¹²⁵I]GIP to LGIPR2 cells in aconcentration-dependent manner (FIG. 3), with an IC₅₀ of 7 mM for GIP(n=5) and 200 for ANTGIP (n=4). Binding of [¹²⁵I]GLP-1 to its βTC3 cellreceptor was displaced fully by GLP-1, but negligibly by ANTGIP, with anIC₅₀ of 4 nM and 80 μM, respectively (n=7; FIG. 3).

Example 3 Intravenous Infusion of Peptides in Fasting Anesthetized Rats

Adult male Sprague-Dawley rats (250-350 g) were purchased from CharlesRiver Co. (Kingston, Mass.). For infusion studies, rats were fastedovernight and then anesthetized using intraperitoneal sodiumpentobarbital. The right jugular vein was cannulated with siliconpolymer, tubing (0.025 in I.D., 0.047 in O.D., Dow Corning Corporation,Midland, Mich.), as described by Xu and Melethil (21). The tubing wasthen connected to an infusion pump (Harvard Apparatus Co., Inc., Millis,Mass.), and freshly made 0.9% NaCl, 5% glucose, arginine, GIP, or GLP-1(peptides and arginine dissolved in 0.9% NaCl) was infused at a rate of0.1 ml/min. Blood (0.5 ml each) was obtained at 0, 10, 20, and 30 min bytranslumbar vena cava puncture, as described by Winsett et al. (1985,Am. J. Physiol., 249:G145-146), and samples were centrifuged at 2,000 gfor 10 min. Serum samples were separated and stored at −20° C. untilassayed for insulin using a radioimmunoassay kit (ICN Biochemicals,Costa Mesa, Calif.), and glucose, using a One Touch Iiβ glucose meter(Lifescan, INS., Milpitas, Calif.).

To examine the insulinotropic effect of GIP in vivo, fasted anesthetizedrats were perfused continuously with three different concentrations ofGIP (0.5, 1.0, and 1.5 nmol/kg) at a rate of 0.1 ml/min for 30 min (10⁻⁸M equivalent to 1 nmol/kg/30 min). Significant increases in plasmainsulin levels were first detected at 15 min, and after completion ofthe GIP infusion, insulin levels were elevated with all three GIPconcentrations (43.5±2.7, 61.6±4.2, and 72.4±3.5 μIU/ml, respectively)compared to control (32.2±3.3 μIU/m, p<0.05, FIG. 4). The concomitantadministration of ANTGIP (100 nmol/kg) completely abolished theinsulinotropic properties of GIP (1.5 nmol/kg), with plasma insulinreturning to control values (FIG. 4). GIP was infused at 0.5, 1.0, and1.5 nmol/kg, with the largest insulin stimulatory response seen with 1.5nmol/kg. ANTGIP (100 nmol/kg) administered concomitantly with GIP 1.5nmol/kg completely abolished its insulinotropic effect, whereas ANTGIPand 0.9% NaCl infusion had no effect on insulin secretion (n=6 for eachgroup, *p<0.05, compared with basal levels).

To examine whether ANTGIP exerted a nonspecific effect on β-cellfunction, GLP-1 (0.4 nmol/kg), glucose (0.8 g/kg), or arginine (375mg/kg) was infused, in the presence or absence of the antagonist for 30min, as described by Wang et al. (13). FIG. 5 shows plasma insulinconcentrations (±SE) in fasted anesthetized rats after a 30-min infusionof GLP-1 (0.4 nmol/kg), glucose (0.8 g/kg), or arginine (375 mg/kg) with(open bars) or without (solid bars) ANTGIP (100 nmol per kg) (n=6 foreach group, *p<0.05, compared with basal levels). GLP-1, glucose, andarginine alone each significantly increased insulin levels after 15 minof infusion, and by 30 min, the insulin levels in GLP-1-, glucose-, andarginine-infused rats were 50.3±3.7, 63.1±2.5, 69.7±5.8 μIU/mlrespectively (p<0.01, compared with control rats, 29.1±2.9 μIU/ml, FIG.5). No significant change in the insulin response was detected whenANTGIP was administered concomitantly (FIG. 5).

Example 4 Insulinotropic Effect of GIP in Trained Conscious Fed Rats

Postprandial plasma insulin and serum glucose levels were studied inconscious trained rats. Previous reports have indicated that the stressresponse to injection in untrained rats might alter their feeding andsubsequently glucose and insulin levels (13). To avoid such a response,rats were trained for 10 d before experimentation. They were fasted from17:00 to 08:00, and 0.9% NaCl (0.3 ml) was injected subcutaneously at08:00 before feeding. After the injection of 0.9% NaCl, animals weregiven rat chow for 30 min, after which it was removed. At the end oftendays, the rats were accustomed to the injection and ate quickly(consuming 4-6 g of rat chow within 30 min).

On the day of the experiment, after fasting from 17:00 the night before,trained rats were injected subcutaneously at 08:00 with 0.3 ml of either0.9% NaCl or ANTGIP (100 nmol/kg). This dose was chosen to approximatelythe amount of peptide used in the anesthetized animal studies of Example3. After injection, six of the fasted control rats were killed to obtainbaseline serum glucose and insulin levels. ANTGIP- or 0.9% NaCl-treatedrats (n=6 in each group) were exposed to chow for 30 min, after whichfood was withdrawn. Rats were then anesthetized by intraperitonealsodium pentobarbital, and blood was collected by translumber vena cavapuncture at 20 and 40 min for the subsequent measurement of plasmainsulin, glucose, and GLP-1.

FIG. 6 shows postprandial plasma insulin and serum glucose levels (±SE)in conscious trained rats (* p<0.01 compared to ANTGIP injection). Inresponse to consuming chow, serum glucose and plasma insulin levelsincreased significantly, with insulin levels of 38.7±5.3 and 58.9±3.7μIU/ml at 20 and 40 min, respectively (p<0.05, FIG. 6A). These increasesin plasma insulin level were nearly abolished by ANTGIP pretreatment; at20 and 40 min, the plasma insulin concentrations were 25.3±4.7 and27.1±2.6 μIU/ml, respectively (p<0.01). Postprandial serum glucoseconcentrations were similar in both saline- and ANTGIP-treated rats(FIG. 6B). To determine whether the effects of the GIP receptorantagonist were mediated through changes in GLP-1 release into thecirculation, postprandial serum GLP-1 levels were measured in bothcontrol and ANTGIP-treated animals. Meal-stimulated serum GLP-1concentrations were not affected by ANTGIP administration. Following theingestion of rat chow, serum GLP-1 levels at 20 min were 280±20 and290±10 pg/ml in control and ANTGIP-treated rats, respectively; at 40min, serum GLP-1 concentrations were 320±10 and 330±20 pg/mgl,respectively.

Example 5 Effect of ANTGIP on Glucose Tolerance and Plasma InsulinLevels

Oral glucose tolerance tests were performed on rats injectedintraperitoneally with ANTGIP (300 ng/kg) or 0.9% saline solution. Afterthe intraperitoneal injection of 0.9% NaCl or ANTGIP, an oral glucosetolerance test was performed. The test was done by administering a 40%glucose solution by oral gavage at a dose of 1 g per kg. The volumeadministered to each rat was approximately 0.5 ml. Blood was obtained atvarious time points for subsequent measurement of plasma insulin andglucose levels.

As expected in view of the experiment in Example 4, rats treated withANTGIP showed reduced the plasma insulin levels (FIG. 7). Surprisingly,plasma glucose was diminished at all time points in rats treated withANTGIP, compared to control rats (FIG. 8). Thus, ANTGIP increasesglucose tolerance, despite its negative effect on the insulinotropicresponse to GIP shown in Examples 3 and 4.

Example 6 Effect of GIP Receptor Antagonist on Intestinal GlucoseAbsorption

Male Sprague-Dawley rats weighing about 200-250 g are fasted overnightand anesthetized using intraperitoneal urethane (about 1.25 g per kgbody weight). After midline laparotomy, an about 30-cm segment ofjejunum, starting at about 5 cm distal to the ligament of Treitz, isisolated and flushed with approximately 20 ml of about 0.9% NaCl. Thejejunal test segments are each perfused twice, initially with controlbuffer and then once again with control buffer or with the testsolution. The test solution consists of Krebs-Ringer-bicarbonate buffercontaining about 5 mmol/L [¹⁴C]D-glucose, and ³H-labeled polyethyleneglycol is included in the luminal perfusate to correct for fluidmovement. The test or control solution is perfused through the jejunalsegment without recirculation at a flow rate of about 1.6 ml/min, usinga Harvard PHD 2000 syringe pump (Harvard Apparatus, Millis, Mass.). Theeffluent from the luminal segment is collected at about 5-min intervalsfor about 30 min. After the initial period of perfusion, the luminalcontents in the jejunum are flushed with about 20 ml of about 0.9% NaClprior to the initiation of the second period of perfusion. In allexperiments, animals are administered either about 0.9% NaCl (control)or ANTGIP (10 nmol/kg body weight) though the inferior vena cava bysingle injection at about time 0 min.

The enclosed FIG. 9 depicts the effects of the GIP receptor antagonist,ANTGIP, on the absorption of free D-glucose from the lumen of thejejunal test segment. Data points are believed to represent the rate ofglucose disappearance from the luminal perfusate corrected for fluidmovement. Results are expressed as the mean ±SE of five experiments.Statistical significance (*) is assigned if P<0.05. As seen in thefigure, a ANTGIP is believed to significantly reduce the absorption ofD-glucose from the jejunal test segment throughout the entire 30-miniperiod of perfusion. Thus, it is believed that one of the mechanisms bywhich GIP receptor antagonism may improve glucose tolerance is bydecreasing intestinal glucose absorption.

For purposes of clarity of understanding, the foregoing invention hasbeen described in some detail by way of illustration and example inconjunction with specific embodiments, although other aspects,advantages and modifications will be apparent to those skilled in theart to which the invention pertains. The foregoing description andexamples are intended to illustrate, but not limit the scope of theinvention. Modifications of the above-described modes for carrying outthe invention that are apparent to persons of skill in medicine,molecular biology, pharmacology, and/or related fields are intended tobe within the scope of the invention, which is limited only by theappended claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference in their entireties to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

1. An antagonist of glucose-dependent insulinotropic polypeptide (GIP)consisting essentially of a 24 amino acid polypeptide corresponding topositions 7-30 of the sequence of GIP.
 2. An isolated polypeptidecomprising an amino acid sequence which specifically interferes with thebiological activity of glucose-dependent insulinotropic polypeptide(GIP) when said polypeptide is administered to an animal in an amounteffective to reduce intestinal uptake of glucose.
 3. A polypeptideaccording to claim 2, wherein said polypeptide comprises an amino acididentical to SEQ ID NO:2.
 4. A polypeptide according to claim 2, whereinthe polypeptide comprises an amino acid sequence at least 95% identicalto SEQ ID NO:2.
 5. A polypeptide according to claim 2, wherein saidpolypeptide comprises an amino acid sequence at least 95% identical toSEQ ID NO:8.
 6. A polypeptide according to claim 2, wherein thepolypeptide comprises an amino acid sequence identical to SEQ ID NO:8.7. A polypeptide according to claim 2, wherein said polypeptidecomprises an amino acid sequence at least 95% identical to SEQ ID NO:3.8. A polypeptide according to claim 2, wherein the polypeptide comprisesan amino acid sequence at least 95% identical to SEQ ID NO:3.
 9. Apolypeptide according to claim 2, wherein said polypeptide comprises anamino acid sequence identical to SEQ ID NO:9.
 10. A polypeptideaccording to claim 2, wherein the polypeptide comprises an amino acidsequence at least 95% identical to SEQ ID NO:9.
 11. A polypeptideaccording to claim 2, wherein said polypeptide comprises an amino acidsequence identical to SEQ ID NO:5.
 12. A polypeptide according to claim2, wherein the polypeptide comprises an amino acid sequence at least 95%identical to SEQ ID NO:5.
 13. A polypeptide according to claim 2,wherein said polypeptide comprises an amino acid sequence identical toSEQ. ID NO
 10. 14. A polypeptide according to claim 2, wherein thepolypeptide comprises an amino acid sequence at least 95% identical toSEQ ID NO:10.
 15. A polypeptide according to claim 2, wherein saidpolypeptide comprises an amino acid sequence identical to SEQ ID NO:13.15. A polypeptide according to claim 2, wherein the polypeptidecomprises an amino acid sequence at least 95% identical to SEQ ID NO:13.17. A polypeptide according to claim 2, wherein said polypeptidecomprises the amino acid sequence of SEQ ID NO:13.
 18. A polypeptideaccording to claim 9, wherein the polypeptide comprises an amino acidsequence at least 95% identical to SEQ ID NO:13.
 19. A polypeptidehaving an amino acid sequence which specifically interferes with thebiological activity of glucose-dependent insulinotropic polypeptide(GIP) when said polypeptide is administered to a mammal in an amounteffective to reduce absorption of glucose from the mammalian gut, saidpolypeptide comprising the amino acid sequence of SEQ ID NO
 6. 20. Apolypeptide according to claim 39, wherein the polypeptide comprises anamino acid sequence at least 95% identical to SEQ ID NO:2.
 21. Anisolated polypeptide antagonist of glucose-dependent insulinotropicpolypeptide (GIP) receptor effective to reduce glucose uptake from amammalian intestine and reduce serum insulin levels.